Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Differentially expressed genes of Acanthamoeba castellanii during encystation

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.     

Atg3-mediated lipidation of Atg8 is involved in encystation of Acanthamoeba

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.     

Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.     

Remarkable Salinity Tolerance of Seven Species of Naked Amoebae (gymnamoebae)

The salinity tolerance of naked amoebae collected from sites ranging from ca. 0‰ to 160‰ were compared in laboratory experiments. Amoebae were collected from hypersaline ponds around the perimeter of the Salton Sea, California, where salinities averaged 160‰, and directly from the shoreline waters of the Sea where salinities were generally between 44 and 48‰. Naked amoebae were also collected from the intertidal zone of a Florida beach, a habitat subject (on occasion) to salinity fluctuations within the range 6–85‰. From these combined sites, 6 clones of amoebae were isolated for salinity tolerance experiments (2 marine beach isolates, 2 Salton Sea isolates, and 2 hypersaline pond isolates). A seventh clone, Acanthamoeba polyphaga, a common freshwater/soil amoeba, was obtained from a Culture Collection. Laboratory experiments compared the effects of gradually changing culture salinity versus no salinity acclimatization. Growth rate and culture yield were used as indices of effect. Generally, amoebae were tolerant over a wide range of salinity conditions (in terms of growth and yield) and were not markedly influenced by pre-conditioning to salinity changes throughout the experiments. Overall, the freshwater amoeba Acanthamoeba grew between 0 and 12‰, the marine clones grew in the range of 2–120‰, and the Salton Sea clones reproduced between 0 and 138 ‰. The hypersaline clones were the most resilient and grew between 0 and 270‰ salt. The survival and activity of large populations of naked amoebae in sites subject to salinity fluctuations suggest that they should be considered in future studies to better understand their, as yet, undefined ecological role.     

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila'', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila'' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.     

Characterization of chitin synthases from Entamoeba

A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine. Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases. CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba. In this study, the primary structures of two putative E. histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E. dispar and the reptilian parasite E. invadens. The latter constitutes the widely used model organism for the study of Entamoeba cyst development. The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity. Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs. 114 kD) as well as in their isoelectric points (5.04 vs. 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain. Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule. Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E. invadens. The time-courses of expression differed slightly between the two ameba CHS genes, as in contrast to CHS-1 RNA, expression of CHS-2 RNA was more transient and no plateau was observed between 8 and 16 hours of encystation. However, both CHS RNAs were no longer detectable after 48 hours when most of the cells had been transformed into mature cysts.     

Construction of EST Database for Comparative Gene Studies of Acanthamoeba

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).     

Effects of monoamine neuromediators on the growth-related variables of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The monoamine neuromediators serotonin (5-HT), histamine, dopamine (DA), and norepinephrine (NE), added to an Escherichia coli K-12 strain MC 4100 culture upon inoculation, stimulate cell proliferation (determined from CFU formation) and biomass accumulation (monitored nephelometrically) during the late lag phase and the early exponential growth phase. These effects are less significant in the late exponential and stationary phase cultures. According to the concentration dependence of the stimulatory effects, the neuromediators can be classified into two groups: (i) the catecholamines DA and NE, whose effects increase almost linearly with increasing concentrations within the range of 0.1–100 μM, and (ii) histamine and 5-HT, which are characterized by bell-shaped concentration dependence curves with maxima at 0.1 (histamine) and 1 μM (5-HT). On an agar-containing medium, the growing E. coli population includes solitary cells and compact cell groups (microcolonies). In this system, both tested catecholamines exert a relatively weak stimulatory influence that manifests itself as an increase in the number of both solitary cells and cell groups, and occurs at concentrations of 10 μM and higher. In analogy to the culture grown on the liquid medium, 5-HT and histamine are distinguished by nonlinear concentration dependence curves: their effects peak at 0.1 μM (histamine) or 1 μM (5-HT); an increase in the neuromediator concentrations results in a decrease in effects that are enhanced by further increasing the concentrations to the submillimolar range. DA increases the percentage of solitary cells, whereas the other tested amines promote cell group formation. The results are interpreted in terms of specific (probably receptor-dependent) mechanisms of action in the neuromediators involved.     

Recent advances in the biocontrol of Orobanche (broomrape) species

Parasitic broomrapes (Orobanche spp.) are majoruncontrolled weeds in the Mediterranean regions of Europe and the NearEast causing major losses to vegetable, grain legume, and sunflowercrops. Selective herbicides alone cannot provide persistent, season-longcontrol of these parasites, and much methyl bromide is used for theircontrol, where affordable. Thus they are excellent targets forbiocontrol. The recent progress by the COST 816 Orobancheworking group in this area is reviewed herein. Natural infestation bythe fly Phytomyza orobanchia of seed capsules of Orobanchecrenata parasitising faba bean halved Orobanche seedproduction while inundative releases of adults reduced it to 5%of viable seeds. The fungi Fusarium arthrosporioides E4a andF. oxysporum E1d, as well as strains of bacteria were isolatedfrom diseased, juvenile, Orobanche flower stalks. They arepathogenic to O. aegyptiaca, O. crenata and O. ramosaon most vegetable crops. A F. oxysporum f. sp.orthoceras was specifically pathogenic to O. cumana onsunflowers. All were used in various experiments with a modicum ofsuccess. Methods were developed to formulate isolated mycelia, whichcould eventually allow the use of transgenic hypervirulent pathogens inasporogenic (deletion) mutants (as a failsafe against spread).Mycotoxins were also isolated from different Fusarium and otherfungal species that kill Orobanche, and are being consideredfor direct use, or to augment other strategies. All threeFusarium spp. used have been transformed with gusand/or gfp genes allowing tracing their movement in theenvironment, and opening the way to future transformations tohypervirulence.     

Recognition of heterospecific parasitism: Competition between Aphidiid (Aphidius ervi) and Aphelinid (Aphelinus asychis) parasitoids of Aphids (Hymenoptera: Aphidiidae; Aphelinidae)

The solitary parasitoids Aphidius erviHaliday (Hymenoptera: Aphidiidae) and Aphelinus asychisWalker (Hymenoptera: Aphelinidae) attacked but generally did not oviposit in pea aphids parasitized by the other species. Wasps selectively oviposited in unparasitized hosts when given a choice. Host discrimination depended on the recognition of internal cues. Females of A. asychiseither could not recognize or ignored A. ervi'sexternal host marking pheromone. Under most conditions, A. ervisurvived in superparasitized hosts, killing competing A. asychislarvae by physical attack and possibly physiological suppression. The outcome of larval competition was not affected by oviposition sequence or age difference between larvae; A. asychissurvived only when it had substantially completed larval development before the host was superparasitized by A. ervi.It is suggested that competition for host resources incurs a cost, for the winner in terms of reduced size or increased development time and for the loser in terms of lost progeny and searching time. Consequently, heterospecific host discrimination can be functional. Internal, and probably general, cues enable wasps to recognize and avoid oviposition in hosts already parasitized by an unrelated species.     

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.     

Predation potential of three flatworm species (Platyhelminthes: Turbellaria) on mosquitoes (Diptera: Culicidae)

We conducted a field survey for flatworms to select species as potential biological control agents against Aedes aegypti and Culex pipiens (Diptera, Culicidae) breeding in artificial containers. Laboratory experiments were performed to determine the daily predation rate, differential predation on each mosquito larval instar, selective predation on either A. aegypti or C. pipiens, and predator tolerance to water from artificial containers. Girardia anceps (Tricladida, Paludicola, Dugesiidae), Mesostoma ehrenbergii and Bothromesostoma cf. evelinae (Rhabdocoela, Typhloplanoida, Typhloplanidae) were found in temporary puddles and permanent pools. In the laboratory, they killed between 52% and 100% of immature mosquitoes coexisting in the same habitat. No preference of flatworms for mosquito preys was detected. Predation rate was related to predator size and instar of preys. Girardia anceps and B. evelinae survived after a dry period and when re-flooding occurred, they laid eggs. Tolerance to water from artificial containers was highest in G. anceps and this species could be a suitable predator to reduce mosquito populations from artificial containers using an inoculative approach.     

Is Acanthamoeba pathogenicity associated with intracellular bacteria?

In addition to the possible role of Acanthamoeba as an evolutionary precursor of pathogenicity in microbial pathogens, it has been suggested that intracellular bacteria or other microbial endosymbionts may also enhance the pathogenicity of Acanthamoeba. Using transmission electron microscopy, polymerase chain reaction and simple culturing, our findings did not reveal any apparent evidence of microbial presence intracellularly of a recently recovered clinical isolate of Acanthamoeba. Based on these findings, it is tempting to speculate that the virulence of Acanthamoeba may not be attributed to the pathogenicity of the endosymbiont alone.     

Calospatha subsumed in Calamus (Arecaceae: Calamoideae)

Summary  Based on previously published phylogenetic research, the genus Calospatha Becc. (Calamoideae) is placed in synonymy within Calamus L. The new combination, Calamus calospathus (Ridl.) W. J. Baker & J. Dransf. is made.     

The Bionomics ofCoptera Haywardi(Ogloblin) (Hymenoptera: Diapriidae) and Other Pupal Parasitoids of Tephritid Fruit Flies (Diptera)

The endoparasitoidCoptera haywardi(Ogloblin) (Diapriidae) was discovered in Mexico attacking the pupae of the Mexican fruit fly,Anastrepha ludens(Loew). Typically, parasitoids of Diptera Cychlorrhapha pupae develop as ectoparasitoids and are generalists that attack hosts in a number of families. Aspects of the bionomics ofC. haywardiwere compared to those of two chalcidoid ectoparasitoids,Dirhinus himalayanusWestwood andSpalangia geminaBoucek.C. haywardideveloped in three genera of Tephritidae, but not in species of other families. The two species of chalcidoids developed in all the calypterate and acalypterate hosts to which they were exposed. In an olfactometerC. haywardipreferredAnastrepha suspensa(Loew) pupae, while the chalcidoids preferred the pupae ofMusca domestica L.This preference inS. geminawas diminished in insects that had been reared onA. suspensa. C. haywardioviposited in theA. suspensapupae that had been previously parasitized by the braconidDiachasmimorpha longicaudata(Ashmead). However, it completed development only in unparasitized pupae. Mortality of the primary parasitoid due toD. himalayanuswas approximately two-thirds the mortality inflicted on the host fly.S. geminadid not discriminate between parasitized and unparasitized pupae ofA. suspensaand developed in both.C. haywardiappears to have a more restricted host range relative to chalcidoid pupal parasitoids and this may be due to its endoparasitic development.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.